Method for improving growth of corynebacterium glutamicum under high temperature stress

Abstract

The invention discloses a method for improving growth of corynebacterium glutamicum under high-temperature stress, and aims to overcome the problems of abnormal growth and metabolism, even death and the like caused by temperature rise of existing microorganisms in an industrial fermentation production process, adaptive laboratory evolution is adopted, corynebacterium glutamicum ATCC 13032 is used as an original strain, and the corynebacterium glutamicum ATCC 13032 is used as an original strain. And carrying out heat stress subculture for 42.9 days, and separating out the strains HES1 and HES2 with improved high temperature resistance. The whole genome is re-sequenced to obtain mutant elements cspB and gltA which enable the strain to generate high-temperature adaptive change. And carrying out high-temperature test on the abrupt change element. In specific implementation, the two mutants are respectively overexpressed, and in a test, compared with a control strain WT, the 42-DEG C high-temperature tolerance is respectively improved by about 14.4% and 7%. Therefore, the beneficial mutant obtained through adaptive laboratory evolution lays a good foundation for application of corynebacterium glutamicum in industrial high-temperature conditions.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and specifically relates to mining element mutants with enhanced tolerance to high temperature stress of Corynebacterium glutamicum through adaptive laboratory evolution and whole genome sequencing. Background Art

[0002] Temperature changes in the environment are one of the most important factors affecting microbial growth, which is specifically reflected in enzyme activity, stability of biomacromolecules, and fluidity and integrity of cell membranes. Most industrial microorganisms are sensitive to temperature. During the process of industrial fermentation of microorganisms, heat is released and the temperature rises, which affects their normal growth and metabolism, and even causes death, which is not conducive to industrial production. At the same time, extreme temperatures can also destroy the structure of microbial cell membranes or interfere with the functions of biomacromolecules such as proteins and RNA. Therefore, in order not to affect the growth and metabolism of microorganisms, reduce the cost of industrial fermentation of microorganisms, and achieve efficient high-temperature fermentation, only by understanding the physiological mechanisms of microbial responses to different environmental stresses and improving the physiological functions of industrial microbial cells can the adaptability of industrial microorganisms to stress in the production environment be significantly improved.

[0003] Adaptive laboratory evolution (ALE) is a commonly used method in biological research, which can provide in-depth understanding of the basic mechanisms of molecular evolution and the adaptive changes accumulated in microbial populations during long-term selection under specific growth conditions. The fully automatic microbial adaptive evolution instrument (EVOL cell) is a microbial domestication equipment developed based on high gas permeability microtubules and single-phase microfluidic technology. It has functions such as microbial cultivation, passaging, chemical factor gradient addition, and oxygen partial pressure control. The instrument uses microtubules as a microbial reactor, which has good permeability to a variety of gases (oxygen, nitrogen, carbon dioxide, etc.), fully meeting the gas exchange requirements of the cultivation process. Using oxygen partial pressure control technology, the gas environment around the reactor can be flexibly controlled, so it can be applied to the cultivation and adaptive evolution of a variety of microorganisms.

[0004] In the field of industrial fermentation, Corynebacterium glutamicum can often provide potential applicable benefits to industrial fermentation when cultivated at higher temperatures, such as reducing fermentation cooling costs, reducing the consumption of water and electricity resources, optimizing the fermentation process, and reducing the risk of pollution during the fermentation process. In addition, in some cases, the fermentation temperature that rises can lead to faster microbial growth and increase overall biomass output, thereby making it an attractive characteristic to improve the high temperature tolerance of the strain industrially. At present, the research on the high temperature resistance of Corynebacterium glutamicum is less, and the regulatory mechanism under heat stress is not yet understood in depth, and the transcriptional response to heat shock and its regulatory system has not yet been analyzed at the whole genome level. Therefore, exploring the high temperature resistance regulatory mechanism of Corynebacterium glutamicum, constructing excellent high temperature resistant strains is of great significance to industrial fermentation. The present invention excavates and characterizes the important functional elements involved in the high temperature resistance of the strain, and constructs an efficient strain with high temperature resistance. Summary of the invention

[0005] First, Corynebacterium glutamicum ATCC13032 was selected as the original parent strain, and the thermostable Corynebacterium glutamicum was screened by using the fully automatic microbial adaptive evolution instrument (EVOL cell) under heat stress conditions;

[0006] Second, after heat adaptation, two individual colonies with the best heat tolerance were isolated from the heat stress screening plate and named HES1 and HES2;

[0007] Third, whole genome resequencing of two thermoevolved strains, HES1 and HES2;

[0008] 4. Overexpress the mutant element obtained in step 3 on the pXMJ19 plasmid to construct a recombinant plasmid;

[0009] 5. Compare the OD of the wild strain containing the empty pXMJ19 plasmid and the recombinant plasmid in step 4 600 value, and screen for mutant elements with improved high temperature tolerance.

[0010] Beneficial effects of the present invention:

[0011] Microorganisms are sensitive to temperature. In the process of industrial fermentation production, the increase in temperature will affect their normal growth and metabolism, and even death, resulting in reduced yield, which is not conducive to industrial production. The present invention obtains two heat-resistant bacteria through adaptive laboratory evolution, and adopts the method of genome resequencing to obtain the cold shock protein cspB and citrate synthase gltA, which theoretically demonstrates the important role of the element in the heat resistance of Corynebacterium glutamicum, and experimental verification confirms the application of the element in improving the resistance of Corynebacterium glutamicum to high temperature stress. Compared with the control strain, its 42°C high temperature tolerance is effectively improved by 14.4% and 7%, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is the technical roadmap of the present invention.

[0013] Figure 2 This is the effect of different temperatures on the growth of strains in the present invention.

[0014] Figure 3 These are the results of the EVOL cell adaptive evolution experiment in the present invention.

[0015] Figure 4 This is a growth test of the evolved bacteria HES1 and HES2 of Corynebacterium glutamicum in the present invention and wild bacteria at different temperatures.

[0016] Figure 5 This is a high temperature tolerance growth test of overexpressing the mutant elements cspB and gltA of Corynebacterium glutamicum in the present invention. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings, but it should not be construed as limiting the present invention. The experimental methods used in the examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the examples can be obtained from commercial sources unless otherwise specified.

[0018] Example 1, High temperature stress adaptability laboratory evolution of wild Corynebacterium glutamicum. A fully automatic microbial adaptability evolution instrument (EVOL cell) was used to screen for high temperature resistant Corynebacterium glutamicum. Corynebacterium glutamicum ATCC13032 was selected as the starting strain, comprising the following steps:

[0019] Step 1: The starting strain culture medium was LBHIS medium, and the culture was carried out at 32°C and 210 rpm until the late logarithmic growth period was reached for 24 h. 600 =6.992, inoculum size is 2%;

[0020] Step 2: Inoculate the starting strain into the seed culture medium and culture for 16 h. 600 The value is transferred to the fermentation medium, such as Figure 2 The cells were cultured at different temperatures for 16 h and the OD 600 value;

[0021] Step 3: EVOL cell acclimation experiment. Inoculate the starting strain into the seed culture medium, culture for 16 hours, dilute with 2% inoculum, transfer 8mL of bacterial solution into the EVOL cell, set the experimental parameters, and start the experiment. Figure 3 As shown, the experimental parameters (temperature, subculture time) were adjusted at any time according to the growth of the strain during the process. After the acclimation, the bacterial solution was extracted and coated on the plate for cultivation;

[0022] Step 4: Determine the growth of the strain at different temperatures, such as Figure 4 As shown, HES1 and HES2 showed significantly improved tolerance to high temperature compared with the wild-type strain. In particular, at 42°C, the biomass of HES1 and HES2 was approximately 2.5 times that of WT.

[0023] Example 2: Construction of recombinant plasmids with mutant elements cspB and gltA obtained by whole genome resequencing. The present invention obtains cspB and gltA mutant elements by whole genome resequencing analysis of the thermotolerant strains HES1 and HES2 that have undergone adaptive laboratory evolution in Example 1, and overexpresses them on the pXMJ19 plasmid to construct a recombinant plasmid. The steps include:

[0024] Step 1: Using high-fidelity DNA polymerase, the obtained HES1 and HES2 genomes and pXMJ19 plasmid as templates, designing upstream and downstream primers of the target gene and pXMJ19, using PCR to obtain mutant target gene fragments and plasmid backbones, and using GoldenGate to connect the pXMJ19 plasmid backbone and the target gene to obtain recombinant expression plasmids containing different mutants of the target coding gene;

[0025] Step 2, perform PCR reaction. The PCR system is: 10 μL of 5×Phusion HF buffer, 0.5 μM of upstream primer P1, 0.5 μM of upstream primer P2, 200 μM of dNTPs, 3% DMSO, 50 ng of template DNA, 0.5 μL of Phusion High-Fidelity DNA polymerase, and add sterile water to make up to 50 μL.

[0026] The PCR reaction procedure is as follows: pre-denaturation at 98°C for 1 minute; denaturation at 98°C for 10 seconds; annealing at 62°C for 20 seconds; extension at 72°C for 3 minutes, 35 cycles; extension at 72°C for 5 minutes, and storage at 4°C.

[0027] The GoldenGate connection system is: 1.5 μL T4 DNA Legase buffer, 1 μL T4 DNA Legase, 1 μL BsaI, 1.5 μL 10×BSA, plasmid backbone, fragment (molar ratio of 1:1, 10 μL in total), and the total volume is 15 μL.

[0028] The PCR connection conditions are: 37°C for 3 min, 22°C for 4 min (40 cycles); 22°C for 30 min (1 cycle); 80°C for 5 min (1 cycle); 4°C for 10 min (1 cycle).

[0029] Step 3: The plasmid library obtained above was introduced into E. coli DH5α competent cells according to the E. coli heat shock transformation method.

[0030] The plasmid backbone primers:

[0031] pXMJ19-F: 5'-CACCAGGTCTCACTAAGGATCCCCGGGTACCGAGC-3'

[0032] pXMJ19-R: 5'-CACCAGGTCTCAAAGCTTAATTAATTCTGTTTCCTGTGT GAAATTGTTA-3'

[0033] The mutant target gene primers are:

[0034] cspB-F-BsaI: 5'-CACTACTGGTCTCAGCTTCTTTAAGAAGGAGATATACATGTGCCTGTCGGAACAGTGAAGTG-3'

[0035] cspB-R-BsaI: 5'-CACTACTGGTCTCAATCCTTAAGACTCAAGCTCCTTCG CAACAACGC-3'

[0036] gltA-F-BsaI

[0037] 5'-CACTACTGGTCTCAAAGCTTCTTTAAGAAGGAGATATACATATGTT TGAAAGGGATATCG-3'

[0038] gltA-R-BsaI

[0039] 5'-CACTACTGGTCTCAATCCTTAGCGCTCCTCGCGAGGAA-3'

[0040] Embodiment 3, as Figure 5 As shown, a high temperature tolerance growth test of overexpressing the mutant elements cspB and gltA of Corynebacterium glutamicum is performed. The following steps are included:

[0041] Step 1, introducing the constructed recombinant plasmid into wild Corynebacterium glutamicum by electroporation;

[0042] Step 2: The recombinant strain was transferred to a shake flask and activated for 20 h. The bacterial solution was transferred to a shake flask containing 20 ml of LBHIS medium. The initial OD 600 The value is 0.1. 600=0.4, add 50 μmol / L IPTG, and perform a growth test at 42°C for 20 h. 600 value.

[0043] like Figure 4 As shown, compared with the control strain (Corynebacterium glutamicum containing the empty plasmid pXMJ19), the high temperature tolerance of cspB and gltA at 42°C was increased by about 14.4% and 7%, respectively. Therefore, cspB-A53G and gltA-R310H are functional elements for improving high temperature tolerance.

[0044] As the main bacterial species for large-scale industrial production of amino acids, Corynebacterium glutamicum has great potential to surpass other industrial microorganisms. Amino acids are widely used in the pharmaceutical industry, infant food, fortified food, health food, chemical industry, animal feed additives and cosmetics industry.

[0045] Based on the genome annotation of Corynebacterium glutamicum, cspB encodes a cold shock-like protein that can regulate the expression of cold stress genes. Corynebacterium glutamicum can produce L-lysine from raw corn starch using the cspB promoter. In addition, the cspB promoter can also enable Corynebacterium glutamicum to effectively produce γ-aminobutyric acid. gltA encodes citrate synthase, which is one of the key enzymes in the tricarboxylic acid cycle and the glyoxylate cycle. The tricarboxylic acid cycle is a central metabolic pathway with three major metabolic functions: supplying biosynthetic precursors, generating energy, and reducing energy when glycolysis cannot meet cellular needs. gltA can also participate in the synthesis pathways of some products, and it participates in the biosynthesis of lysine in Corynebacterium glutamicum together with the pck, pgi and hom genes. gltA has also been identified as the most critical gene for the production of O-acetylhomoserine (OAH). By overexpressing gltA, the succinic acid production of Corynebacterium glutamicum can be increased and the pyruvic acid production can be reduced.

Claims

1. A method for improving the growth of Corynebacterium glutamicum under high temperature stress, characterized in that: The following steps are involved: Step 1, selecting Corynebacterium glutamicum ATCC13032 as the original parent strain, and screening thermostable Corynebacterium glutamicum by using a fully automatic microbial adaptive evolution instrument (EVOL cell) under heat stress conditions; Step 2: After heat adaptation, two single colonies with the best heat resistance were isolated from the heat stress screening plate and named HES1 and HES2, and the growth of the strains at different temperatures was measured; Step 3, perform whole genome resequencing of the thermally evolved strains HES1 and HES2; Step 4, overexpressing the mutant elements cspB and gltA obtained in step 3 on the pXMJ19 plasmid to construct a recombinant plasmid; Step 5: Compare the OD of the wild strain containing the empty pXMJ19 plasmid with the recombinant plasmid in step 4 600 Value, verify the mutation elements that improve high temperature tolerance.

2. A method for improving the growth of Corynebacterium glutamicum under high temperature stress according to claim 1, characterized in that: In the step 1, the starting strain culture medium is LBHIS culture medium. The above method was cultured at 32°C and 210 rpm for 24 h to reach the late logarithmic growth stage, and the OD 600 =6.

992. The starting strain was inoculated into the seed culture medium at an inoculation volume of 2% and cultured for 16 h. 600 The values ​​were transferred to the fermentation medium and cultured at different temperatures for 16 h. The OD 600 value. The starting strain was inoculated, cultured for 16 h, diluted with 2% inoculum, 8 mL of bacterial solution was transferred into EVOL cell, experimental parameters were set, and the experiment was started. During the experiment, the experimental parameters (temperature, subculture time) were adjusted at any time according to the growth of the strain. After the acclimation, the bacterial solution was extracted and coated on a plate for cultivation.

3. A method for improving the growth of Corynebacterium glutamicum under high temperature stress according to claim 1, characterized in that: In step 2, the temperature gradient is 30°C, 32°C, 35°C, 37°C, 40°C, 42°C and 43°C. Compared with the wild-type strain, the tolerance of HES1 and HES2 to high temperatures above 40°C is significantly improved. As mentioned above, at 42°C, the biomass of HES1 and HES2 was about 2.5 times that of WT.

4. A method for improving the growth of Corynebacterium glutamicum under high temperature stress according to claim 1, characterized in that: In the step 3, the thermotolerant strains HES1 and HES2 obtained by adaptive laboratory evolution in claim 2 are subjected to whole genome resequencing analysis to obtain cold shock protein cspB and citrate synthase gltA mutation elements. The cspB, relative to the amino acid sequence shown in SEQ ID No.2, has only a mutation from alanine A at position 53 to glycine G. More specifically, its amino acid sequence is shown in SEQ ID No.

6. The gltA, relative to the amino acid sequence shown in SEQ ID No.4, has only the 310th arginine R mutated to histidine H. More specifically, its amino acid sequence is shown in SEQ ID No.

8.

5. The gene encoding the cold shock protein cspB mutant according to claim 4, preferably, the nucleotide sequence thereof is shown as SEQ ID No.

5.

6. The gene encoding the citrate synthase gltA mutant as claimed in claim 4, preferably, the nucleotide sequence of which is shown in SEQ ID No.

7.

7. A method for improving the growth of Corynebacterium glutamicum under high temperature stress according to claim 1, characterized in that: In the step 4, the coding genes as described in claim 4 are contained and overexpressed on the pXMJ19 plasmid to construct a recombinant plasmid. The method adopts high-Phusion High-Fidelity DNA polymerase, takes the obtained HES1 and HES2 genomes and pXMJ19 plasmid as templates, and designs upstream and downstream primers of the target gene and pXMJ19. The mutant target gene fragment and plasmid skeleton are obtained by PCR, and GoldenGate is used to connect the pXMJ19 plasmid skeleton and the target gene to obtain a recombinant expression plasmid containing different mutants of the target coding gene. The PCR system is: 10 μL of 5×Phusion HF buffer, 0.5 μM of upstream primer P1, 0.5 μM of upstream primer P2, 200 μM of dNTPs, 3% DMSO, 50 ng of template DNA, 0.5 μL of Phusion High-Fidelity DNA polymerase, and sterile water is added to make up to 50 μL. The PCR reaction procedure is as follows: pre-denaturation at 98°C for 1 min; denaturation at 98°C for 10 s; and annealing at 62°C for 20 s. The cells were extended at 72°C for 3 min and cycled 35 times; the cells were then extended at 72°C for 5 min and stored at 4°C. The GoldenGate connection system is: 1.5 μL T4 DNA Legase buffer, 1 μL T4 DNA Legase, 1 μL BsaI, 1.5 μL 10×BSA, plasmid backbone, fragment (molar ratio of 1:1, 10 μL in total), and the total volume is 15 μL. The PCR connection conditions are: 37°C for 3 min, 22°C for 4 min (40 cycles); 22°C for 30 min (1 cycle); 80°C for 5 min (1 cycle); 4°C for 10 min (1 cycle). The plasmid library obtained above is introduced into Escherichia coli DH5α competent cells according to the Escherichia coli heat shock transformation method. The plasmid backbone primers: pXMJ19-F: 5'-CACCAGGTCTCACTAAGGATCCCCGGGTACCGAGC-3' pXMJ19-R: 5'-CACCAGGTCTCAAAGCTTAATTAATTCTGTTTCCTGTGTGAAATTGTTA-3' The mutant target gene primers are: cspB-F-BsaI: 5'-CACTACTGGTCTCAGCTTCTTTAAGAAGGAGATATACATGTGCCTGTCGGAACAGTGAAGTG-3' cspB-R-BsaI: 5'-CACTACTGGTCTCAATCCTTAAGACTCAAGCTCCTTCGCAACAACGC-3' gltA-F-BsaI 5'-CACTACTGGTCTCAAAGCTTCTTTAAGAAGGAGATATACATATGTTTGAAAGGGATATCG-3' gltA-R-BsaI 5'-CACTACTGGTCTCAATCCTTAGCGCTCCTCGCGAGGAA-3' 8. A method for improving the growth of Corynebacterium glutamicum under high temperature stress according to claim 1, characterized in that: The step 5 comprises the use of the coding gene as claimed in claim 4 in enhancing the high temperature tolerance of the recombinant bacteria of Corynebacterium glutamicum. In particular, the high temperature tolerance growth test of overexpressing the mutant elements cspB and gltA of Corynebacterium glutamicum. The constructed recombinant plasmid is introduced into wild Corynebacterium glutamicum by the electrotransformation method. The recombinant strain was inoculated into a shake flask and activated for 20 h. The bacterial solution was transferred into a shake flask containing 20 ml of LBHIS medium. The initial OD 600 The value is 0.

1. The OD 600 =0.4, add 50 μmol / L IPTG, and perform a growth test at 42°C for 20 h. 600 value. Compared with the control strain (Corynebacterium glutamicum containing the pXMJ19 empty plasmid), the high temperature tolerance of the cspB and gltA at 42°C is increased by about 14.4% and 7% respectively.

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